Role of the C8 gem-dimethyl group of bryostatin 1 on its unique pattern of biological activity

Article abstract of DOI:10.1016/j.bmcl.2012.04.073

The role of the C₈ gem-dimethyl group in the A-ring of bryostatin 1 has been examined through chemical synthesis and biological evaluation of a new analogue. Assays for biological function using U937, K562, and MV4-11 cells as well as the profiles for downregulation of PKC isozymes revealed that the presence of this group is not a critical determinant for the unique pattern of biological activity of bryostatin.

Full text of DOI:10.1016/j.bmcl.2012.04.073

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4084–4088
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Role of the C8 gem-dimethyl group of bryostatin 1 on its unique pattern
of biological activity
Gary E. Keck a, , Yam B. Poudel a, Arnab Rudra a, Jeffrey C. Stephens a, Noemi Kedei b, Nancy E. Lewin b,
⇑
Peter M. Blumberg b
a University of Utah, Department of Chemistry, 315 South 1400 East, RM 2020, Salt Lake City, UT 84112, USA
b Laboratory of Cancer Biology and Genetics, Center for Cancer Research, NCI, NIH, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The role of the C8 gem-dimethyl group in the A-ring of bryostatin 1 has been examined through chemical
synthesis and biological evaluation of a new analogue. Assays for biological function using U937, K562,
and MV4-11 cells as well as the profiles for downregulation of PKC isozymes revealed that the presence
of this group is not a critical determinant for the unique pattern of biological activity of bryostatin.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 2 February 2012
Revised 12 April 2012
Accepted 16 April 2012
Available online 24 April 2012
Keywords:
Bryostatin
Phorbol ester
Protein kinase C
Cancer
Alzheimer’s disease
Bryostatin 1 is the most thoroughly studied member of a family
of over 20 complex macrolide natural products originally isolated
by Pettit and coworkers from the marine bryozoan Bugula neritina.1
It was later discovered that the bacterial endosymbiont Candidatus
endobugula sertula was the true source of the bryostatins.2 The bry-
ostatins are composed of three highly functionalized pyran rings
embedded in a macrocycle and differ primarily in substitution at
the C7 and C20 positions.3 Bryostatin 1 has been found to display
a remarkably wide range of biological activity. Due to its initially
promising anticancer activity, bryostatin 1 has been used in
numerous phase I and II clinical trials for cancer chemotherapy.
Moreover, bryostatin 1 has been shown to reverse multidrug resis-
tance4 as well as to synergize5 with other well known oncolytic
drugs such as paclitaxel, vincristine and cisplatin. However, unlike
most anticancer drugs, bryostatin 1 stimulates the immune sys-
tem, a property that has recently been utilized to overcome HIV la-
tency in lymphocytes.6 Additionally, bryostatin 1 has displayed
promising neurologic effects. Bryostatin 1 has also been shown to
enhance memory and learning in animal models7,8 and has shown
activity against Alzheimer’s disease in transgenic mice. Moreover,
it has recently been reported that bryostatin 1 is able to stimulate
repair of the neural damage and reestablishment of synapses for up
to 24 h after stroke in rats.9
The mechanism by which bryostatin 1 evokes these diverse bio-
logical effects is believed to arise largely from the modulation of
protein kinase C isozymes (PKCs) and other C1 domain containing
proteins upon their binding of bryostatin 1.10 Due to their impor-
tant role in signaling, PKCs have emerged as an attractive target
in drug discovery.11 Several natural products such as phorbol es-
ters, bryostatins, indolactams, and aplysiatoxin are known high
affinity exogenous ligands for PKCs. An important distinction
among various PKC ligands is that, although they bind to the same
C1 domain of PKCs, the biological responses subsequent to bind-
ings can be very different. For example phorbol esters such as
phorbol 12-myristate-13-acetate (PMA) and bryostatin 1 bind to
the same C1 domain of PKC with high affinity; however, PMA is tu-
mor promoting whereas bryostatin 1 is not.12 In addition, bryosta-
tin 1 antagonizes many of the PMA induced response which it does
not induce itself.
Our group is endeavoring to understand the structural features
of bryostatin 1 responsible for its unique biological activity. To this
end, we have synthesized and analyzed various bryopyran struc-
tures13 using the pyran annulations methodology14 developed in
our laboratories that has proven critical in recently reported total
syntheses of bryostatins.15 Representative examples of our bryopy-
ran analogues are shown in Figure 1. Examination of analogue
Merle 23 revealed that substitution on A- and B-rings is critical
for obtaining antagonism of phorbol ester induced biological re-
sponses. The roles of individual substituents in this region includ-
ing the C30 carbomethoxy group (Merle 28), C9 hydroxy group
⇑
Corresponding author. Tel.: +1 801 581 7055; fax: +1 801 585 0024.
E-mail address: keck@chem.utah.edu (G.E. Keck).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.073

G. E. Keck et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4084–4088
4085
and provided the corresponding seco acid. Although both the C16
–
C17 olefin and C20 ketone proved unreactive towards m-CPBA, it
was necessary to stop the reaction after three and half hours in or-
der to prevent the epoxidation of the C13–C30 olefin. Yamaguchi
macrolactonization of the seco acid then furnished the macrolac-
tone 2 in excellent yield.17 To preclude competing enolization of
the C7 acetate in the ensuing aldol reaction,13c,d the C7 acetate
was removed using K2CO3/MeOH (without interference from the
macrolactone ester functionality) and the resulting C7 alcohol
was protected as the TES ether. The aldol reaction of the C-ring ke-
tone 3 with freshly distilled methyl glyoxylate using LDA then pro-
vided the corresponding aldol adduct as
a
mixture of
diastereomers which were subjected to elimination by stirring
with acetic anhydride. It proved necessary to keep the reaction at
room temperature in order to avoid deprotection of the C7 TES
group. This reaction provided the desired
as a single isomer.
a,b-unsaturated ester 4
Luche reduction of the C20 ketone provided the alcohol which
was found to be unstable for isolation and purification purposes
(Scheme 2). Thus, this intermediate was immediately subjected
to esterification by reaction of the crude alcohol with 2,4-octadie-
noic anhydride. The C7 alcohol was then revealed by removal of the
TES group by reaction with PPTS in MeOH.
Oxidation of the alcohol to the corresponding ketone proved
uneventful using the Dess-Martin oxidation.18 However, subse-
quent conversion of the ketone 6 to the desired alkene proved to
be a daunting task. Attempted olefination of the ketone using Peta-
sis reaction conditions provided a complex mixture of products.19
On the other hand, we were initially reluctant to use the Wittig
reaction20 on such a complex substrate due to the following con-
cerns: (i) low reactivity of the sterically hindered C7 ketone to-
wards the Wittig reagent, leading to enolization, (ii) potential b-
elimination of the C3-OBPS group, and (iii) migration of the C-ring
olefin to an internal position. The latter two processes had been ob-
served in our laboratory during the synthesis of related structures.
However, when the ketone 7 was subjected to Wittig reaction
Figure 1. Structure of bryostatin 1 and bryopyrans.
(Merle 30) and C7 acetate group (Merle 27) have been addressed,
revealing that none of these substituents alone acts as a functional
switch for PMA versus bryostatin like activity. Herein we examine
the role of the final such substituent, the C8 gem-dimethyl group,
through chemical synthesis and biological evaluation of the new
bryopyran analogue Merle 32.16
The synthesis of Merle 32 commenced from intermediate 1
which had been prepared previously enroute to analogues Merle
28 and Merle 30 (Scheme 1). The C25 alcohol was first freed by re-
moval of the PMB group using DDQ. Subjection of the resulting hy-
droxy-thioester to oxidative hydrolysis using m-CPBA in aqueous
THF selectively cleaved the thioester in the presence of the C7 ester
Scheme 1. Preparation of macrolactone and further functionalization.
Scheme 2. Completion of the C8 gem-dimethyl analogue Merle 32.

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G. E. Keck et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4084–4088
conditions, we were pleasantly surprised to find that the reaction
provided the desired product in excellent yield in just 15 min. Re-
moval of the BPS group followed by global deprotection using LiBF4
then completed the synthesis of Merle 32.21
Merle 32 was also evaluated using the human leukemia cell
lines K56224 and MV4-11,25 which both show distinctive dose
dependent patterns for inhibition of proliferation in the presence
of PMA or bryostatin 1. As can be seen in Figure 3, Merle 32 was
strongly antiproliferative, as was PMA but not bryostatin 1. More-
over Merle 32 did not block the response to PMA, while bryostatin
1 did. These results indicate that the behavior shown by Merle 32 is
not limited to just the U937 cell line and indeed was even more
PMA-like in these other two cell lines. Here Merle 32 showed no
ability to block the effect of 10 nM PMA, while bryostatin 1 was
effective in that regard.
Downregulation of PKC isoforms and other C1 domain con-
taining proteins subsequent to ligand binding is another charac-
teristic phenomenon displayed by PKCs and is also important in
determining their functional response to different ligands.26 Dose
dependent patterns of downregulation were determined for PMA,
bryostatin 1, and Merle 32 in the K562 cells (Fig. 4). Here bryost-
atin 1 and PMA showed distinctly different patterns. Bryostatin 1
The biological evaluation of Merle 32 began by determining its
binding affinity (Ki) towards PKC in vitro.22 Its Ki of 1.08 0.16 nM
proved similar to that of bryostatin 1 (Ki = 1.35 nM) and to other
bryopyran analogues prepared previously. The biological profile
of Merle 32 was initially addressed in the U937 human lymphoma
cell line using proliferation and attachment assays (both measures
of differentiation in this cell line). The U937 cells display differen-
tial response towards tumor-promoting PMA and bryostatin 1.23
Specifically, PMA induces attachment and inhibits the proliferation
of U937 cells whereas bryostatin 1 has little effect. On the other
hand, bryostatin 1 blocks both responses to PMA in a dose depen-
dent manner when the two agents are administered together. It
can be seen (Fig. 2) that Merle 32 induced the attachment of
U937 cells in a manner largely similar to PMA, although the max-
imal attachment was not quite as great. Similar observations were
made in the proliferation assay, but with even closer resemblance
to the PMA response.
was more effective in downregulating PKC-
biphasic downregulation of PKC-d (Fig. 4). Moreover, it did not
cause the prominent induction of PKC- and RasGRP3 observed
a and b and showed
e
Figure 2. Effect of Merle 32 on proliferation and attachment of U937 cells. Cells were treated 24 h after seeding with the indicated concentrations of the different compounds.
The floating and attached cells were counted 60 h after treatment as described earlier (13a). Values represent the mean SEM of five independent experiments.

G. E. Keck et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4084–4088
4087
Figure 3. Effect of Merle 32 on proliferation of K562 and MV4-11 cells. Cells were seeded and treated as described for the U937 cells (13a). Total cell numbers were
determined 60 h after treatment. Values represent the mean SEM of three independent experiments.
in these cells with PMA. Merle 32 showed a pattern largely sim-
ilar to that of PMA but with approximately 10-fold weaker
potency.
responsible for its unique biological activity. None of these
groups was found to function singly as a biological switch, thus
it appears that the unique activity of bryostatin 1 relies on a
more subtle interplay between some combinations of these
groups. It is interesting to note that those analogues with two
polar substituents in the A–B ring region (Merle 28 and 30) give
biological results similar to those for bryostatin 1, while those
with fewer or no polar groups (Merle 32, 27 and 23) are PMA-
like or largely so. Thus, it appears that a proper combination of
polar substituents in the A–B ring region may be needed for
mimicking the activity of bryostatin 1. Efforts to identify suitable
combinations of groups in this region are in progress and will be
reported in due course.
The results from the proliferation assays in the various cell lines
together with these downregulation assays indicate that Merle 32
induces biological responses largely similar to those of PMA rather
than bryostatin 1. If the C8 gem-dimethyl group was to serve as a
functional switch, the biological results displayed by Merle 32 in
these systems would be closer to those of bryostatin 1 than to
those of Merle 23, which differs from Merle 32 only at C8, whereas
they very closely resemble those of Merle 23.
To date, we have studied the role of four individual groups in
the A–B region of bryostatin 1 that have been implicated as

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Mohammad, R. M.; Wall, N. R.; Dutcher, J. A.; Katib, A. M. Clin. Cancer Res. 2000,
6, 4950; (c) Basu, A.; Lazo, J. S. Cancer Res. 1992, 52, 3119.
6. Mehla, R.; Bivalkar-Mehla, S.; Zhang, R.; Handy, I.; Albrecht, H.; Giri, S.;
Nagarkatti, P.; Nagarkatti, M.; Chauhan, A. PLoS One 2010, 5, e11160.
7. (a) Alkon, D. L.; Epstein, H.; Kuzirian, A.; Bennett, M. C.; Nelson, T. J. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 16432; (b) Sun, M.-K.; Alkon, D. L. Eur. J. Pharmacol.
2005, 512, 43.
8. Etcheberrigaray, R.; Tan, M.; Dewachter, I.; Kuiperi, C.; van der Auwera, I.;
Wera, S.; Qiao, L.; Bank, B.; Nelson, T. J.; Kozikowski, A. P.; van Leuven, F.; Alkon,
D. L. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11141.
9. Sun, M.-K.; Hongpaisan, J.; Nelson, T. J.; Alkon, D. L. Proc. Natl. Acad. Sci. U.S.A.
2008, 105, 13620.
10. Reyland, M. E.; Insel, P. A.; Messing, R. O.; Dempsey, E. C.; Newton, A. C.;
Mochly-Rosen, D.; Fields, A. P. Am. J. Physiol. Lung Cell. Mol. Physiol. 2000, 279,
429.
11. Parker, P. J., Dekker, L. V., Eds.; Protein Kinase C; R.G. Landes Publishers:
Georgetown, TX, 1997.
12. Blumberg, P. M.; Pettit, G. R. In New Leads and Targets in Drug Research;
Krosgaard-Larsen, P., Christensen, C. B., Kodof, H., Eds.; Munksgaard:
Copenhagen, 1992; p 273.
13. (a) Keck, G. E.; Kraft, M. B.; Truong, A. P.; Li, W.; Sanchez, C. C.; Kedei, N.; Lewin,
N.; Blumberg, P. M. J. Am. Chem. Soc. 2008, 130, 6660; (b) Keck, G. E.; Li, W.;
Kraft, M. B.; Kedei, N.; Lewin, N. E.; Blumberg, P. M. Org. Lett. 2009, 11, 2277; (c)
Keck, G. E.; Poudel, Y. B.; Welch, D. S.; Kraft, M. B.; Truong, A. P.; Stephens, J. C.;
Kedei, N.; Lewin, N. E.; Blumberg, P. M. Org. Lett. 2009, 11, 593; (d) Keck, G. E.;
Poudel, Y. B.; Rudra, A.; Stephens, J. C.; Kedei, N.; Lewin, N. E.; Peach, M. L.;
Blumberg, P. M. Angew. Chem., Int. Ed. 2010, 49, 4580.
Figure 4. Modulation of C1 domain containing proteins upon 24 h treatment of
K562 cells. K562 cells (4 ml of 150,000 cells/ml) were treated 24 h after plating with
the indicated concentrations of the compounds for 24 h. Western blot analysis was
performed on the total cell lysates as described earlier.27 Actin provides a loading
control. A representative image from three independently performed experiments
is shown.
Acknowledgments
14. (a) Keck, G. E.; Covel, J. A.; Schiff, T.; Yu, T. Org. Lett. 2002, 4, 1189; (b) Keck, G.
E.; Truong, A. P. Org. Lett. 2005, 7, 2149; (c) Keck, G. E.; Truong, A. P. Org. Lett.
2005, 7, 2153.
15. (a) Keck, G. E.; Poudel, Y. B.; Cummins, T. J.; Rudra, A.; Covel, J. A. J. Am. Chem.
Soc. 2011, 133, 744; (b) Wender, P. A.; Schrier, A. J. J. Am. Chem. Soc. 2011, 133,
9228; (c) Lu, Y.; Woo, S. K.; Krische, M. J. J. Am. Chem. Soc. 2011, 133, 13876.
Financial support was provided by the NIH through Grant
GM28961 and through the Intramural Research Program, CCR,
NCI, NIH.
16. For gem-dimethyl containing analogues with
a B-ring acetal moiety, see:
Supplementary data
Wender, P. A.; Baryza, J. L.; Brenner, S. E.; DeChristopher, B. A.; Loy, B. A.;
Schrier, A. J.; Verma, V. A. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 6721.
17. Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull. Chem. Soc. Jpn.
1979, 52, 1989.
18. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
19. Petasis, N. A.; Bzowej, E. I. J. Am. Chem. Soc. 1990, 112, 6392.
20. Wittig, G.; Schollkof, U. Chem. Ber. 1954, 87, 1318.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
04.073.
21. Lipschutz, B. H.; Harvey, D. F. Synth. Commun. 1982, 14, 267.
22. Lewin, N. E.; Blumberg, P. M. Methods Mol. Biol. 2003, 233, 129.
23. Vrana, J. A.; Saunders, A. M.; Srikumar, P. C.; Grant, S. Differentiation 1998, 63,
33.
24. Hocevar, B. A.; Morrow, D. M.; Tykocinski, M. L.; Fields, A. P. J. Cell Sci. 1992,
101, 671.
25. Lange, B.; Valtieri, M.; Santoli, D.; Caracciolo, D.; Mavilio, F.; Gemperlein, I.;
Griffin, C.; Emanuel, B.; Finan, J.; Nowell, P. Blood 1987, 70, 192.
26. Leontieva, O. V.; Black, J. D. J. Biol. Chem. 2004, 279, 5788.
27. Kedei, N.; Telek, A.; Czap, A.; Lubart, E. S.; Czifra, G.; Yang, D.; Chen, J.;
Morrison, T.; Goldsmith, P. K.; Lim, L.; Mannan, P.; Garfield, S. H.; Kraft, M. B.;
Li, W.; Keck, G. E.; Blumberg, P. M. Biochem. Pharmacol. 2011, 81, 1296.
References and notes
1. Pettit, G. R.; Herald, C. L.; Doubek, D. L.; Herald, D. L. J. Am. Chem. Soc. 1982, 104,
6846.
2. Lopanik, N.; Gustafson, K. R.; Lindquist, N. J. Nat. Prod. 2004, 67, 1412.
3. For a recent review of the bryostatins, see: Hale, K. J.; Manaviazar, S. Asian J.
Chem. 2010, 5, 704.
4. Spitaler, M.; Utz, I.; Hilbe, W.; Hofmann, J.; Grunicke, H. H. Biochem. Pharmacol.
1998, 56, 861.
5. (a) Koutcher, J. A.; Motwani, M.; Zakian, K. L.; Li, X.-K.; Matei, C.; Dyke, J. P.;
Ballon, D.; Yoo, H.-H.; Schwartz, G. K. Clin. Cancer Res. 2000, 6, 1498; (b)